It is possible to specifically integrate foreign genes into the plant genome by genetic engineering. This process is referred to as transformation and the resulting plants as transgenic plants. The main objectives are plant protection and an increase in quality of the harvest products. Examples of plant protection measures are: (i) herbicide-tolerant plants (DE-A-3701623; Stalker (1988) Science 242, 419), (ii) insect-resistant plants (Vaek (1987) Plant Cell 5, 159-169), (iii) virus-resistant plants (Powell (1986) Science 232, 738-743) and (vi) ozone-resistant plants (Van Camp (1994) BioTech. 12, 165-168). Examples of increase in quality are: (i) decrease in perishability of fruits (Oeller (1991) Science 254, 437-439), (ii) increase in starch production in potato tubers (Stark (1992) Science 242, 419), (iii) modification in starch (Visser (1991) Mol. Gen. Genet. 225, 289-296) and lipid composition (Voelker (1992) Science 257, 72-74) and (iv) production of polymers foreign to the plant (Poirer (1992) Science 256, 520-523). A prerequisite for producing transgenic plants is the availability of suitable transformation systems and the existence of selectable marker allowing the identification of successfully transformed plant cells.
For the transformation there are presently several methods available. The method most frequently used for transforming dicotyledonous plants is the Agrobacterium-mediated gene transfer. Here, use is made of the natural capability of the soil bacterium of integrating genetic material into the plant genome. Further suitable methods are, e.g., protoplast transformation by polyethylene glycol induced DNA transfer, electroporation, sonication or microinjection as well as the transformation of intact cells or tissues by micro- or macroinjection into tissues or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, vacuum infiltration of seed and the biolistic gene transfer.
Since quite independently of the method of transformation only few cells carry the desired properties, a selectable marker is integrated into the plant genome by conventional methods besides the target gene which allows the identification of transgenic cells. Presently, mainly genes are used for selecting transformed plant cells that mediate a herbicide or antibiotics tolerance. Suitable resistance genes are, e.g., the bar gene from Streptomyces hygroscopicus, which mediates resistance to the total herbicide phosphinothricine (De Block (1987) EMBO J. 6, 2513-2518), or the nptII gene from the transposon Tn5 of Escherichia coli, which confers resistance to the antibiotic kanamycin (Herrera-Estrella (1983) EMBO J. 2, 987-995). Depending on the plant species, the methods mentioned are not always effective and frequently negatively affect plant regeneration. Also, the use of genes mediating antibiotics resistances is undesired in the foodstuff sector. Furthermore, it is necessary to manipulate several enzymatic steps to control complex metabolic processes, i.e., it is essential in the area of xe2x80x9cmetabolic engineeringxe2x80x9d to allow for the possibility of multiple transformations of transgenic plants. The above-mentioned reasons have prompted the intensive search for other selectable markers. Despite intensive efforts only few new markers have been successfully used for selecting transformed plant cells. On the basis of the expression of a mannose-6-phosphate isomerase a positive selection on mannose-containing culture media for transformed plant cells could be established (WO 94/20627). Another process makes use of the capability of a deaminase from Aspergillus terreus to detoxicate the insecticide Blasticidin S (Tamura (1995) Biosci. Biotechnol. Biochem. 59, 2336-2338).
The present invention relates to the use of DNA sequences encoding a protein with the biological activity of a 2-deoxyglucose-6-phosphate (2-DOG-6-P) phosphatase as selection marker in plant cells for selecting transformed plants. Furthermore, the invention relates to recombinant DNA molecules containing such DNA sequences, with the latter being operably linked to regulatory sequences of a promoter active in plants and transcription termination and/or polyadenylation signals. Also, vectors, host cells and kits are described containing such recombinant DNA molecules, as well as plant cells and plants transformed with the recombinant DNA molecules. The present invention furthermore relates to processes for producing transgenic plants which due to the introduction of the above-described recombinant DNA molecules can be selected on media containing 2-deoxyglucose. Finally, the present invention relates to transgenic plants, plant cells and tissues containing the DNA molecule according to the invention or being obtained by the process described above, as well as harvest products and propagation material of the transgenic plants described.